革兰氏阴性细菌蛋白分泌系统研究进展

革兰氏阴性细菌由于具有复杂的双层膜结构,其蛋白质分泌能力较差.这使得革兰氏阴性细菌的典型菌株——大肠杆菌作为最常用的受体细胞在生物制药工程和其他生物技术产品生产中受到一定的限制.因此,革兰氏阴性细菌蛋白分泌系统的研究具有重要意义.本文详细地归纳了革兰氏阴性细菌已知的蛋白分泌系统,分别从分泌系统的分泌过程、分泌蛋白类别、...     

Type I secretion in gram-negative bacteria

In gram-negative bacteria, type I secretion is carried out by a translocator made up of three proteins that span the cell envelope. One of these proteins is a specific outer membrane protein (OMP) and the other two are cytoplasmic membrane proteins: an ATP-binding cassette (ABC) and the so-called membrane fusion or adaptor protein (MFP). Type I secretion is sec-independent and bypasses the periplasm. This widespread pathway allows the secretion of proteins of diverse sizes and functions via a C-terminal uncleaved secretion signal. This C-terminal secretion signal specifically recognizes the ABC protein, triggering the assembly of the functional trans-envelope complex. This report will mainly deal will recent data concerning the structure and assembly of the secretion complex as well as the effects and role of substrate folding on secretion by this pathway.     

Biology of type II secretion

The type II secretion pathway or the main terminal branch of the general secretion pathway, as it has also been referred to, is widely distributed among Proteobacteria, in which it is responsible for the extracellular secretion of toxins and hydrolytic enzymes, many of which contribute to pathogenesis in both plants and animals. Secretion through this pathway differs from most other membrane transport systems, in that its substrates consist of folded proteins. The type II secretion apparatus is composed of at least 12 different gene products that are thought to form a multiprotein complex, which spans the periplasmic compartment and is specifically required for translocation of the secreted proteins across the outer membrane. This pathway shares many features with the type IV pilus biogenesis system, including the ability to assemble a pilus-like structure. This review discusses recent findings on the organization of the secretion apparatus and the role of its various components in secretion. Different models for pilus-mediated secretion through the gated pore in the outer membrane are also presented, as are the possible properties that determine whether a protein is recognized and secreted by the type II pathway.     

小灵猫的体重对其泌香影响的研究

本文就人工饲养条件下的小灵猫体重对其泌香的影响进行了研究。试验结果表明,体重对小灵猫的泌香量影响不大,差异不显著（Ｐ＞００５）。但接近野生小灵猫体重的个体组泌香量稍高,随着体重的增加,小灵猫的泌香量有所减少。因此,控制在人工饲养条件下的小灵猫体重对降低饲养成本,提高产香量有一定的意义     

Conditions leading to secretion of a normally periplasmic protein in Escherichia coli.

The phosphate-binding protein (PhoS) is a periplasmic protein which is part of the high-affinity phosphate transport system of Escherichia coli. Hyperproduction of PhoS in strains carrying a multicopy plasmid containing phoS led to partial secretion of the protein. By 6 h after transfer to phosphate-limiting medium, about 13% of the total newly synthesized PhoS was secreted to the medium. Kinetic studies demonstrated that this secretion consists of newly synthesized PhoS. This secretion occurs in PhoS-hyperproducer strains but not in a PhoS-overproducer strain. Another type of secretion concerning periplasmic PhoS was observed in both PhoS-hyperproducer and PhoS-overproducer strains. This mode of secretion depended upon the addition of phosphate to cells previously grown in phosphate-limiting medium.     

Control of effector export by the Pseudomonas aeruginosa type III secretion proteins PcrG and PcrV

Pseudomonas aeruginosa uses a type III secretion system to inject protein effectors into a targeted host cell. Effector secretion is triggered by host cell contact. How effector secretion is prevented prior to cell contact is not well understood. In all secretion systems studied to date, the needle tip protein is required for controlling effector secretion, but the mechanism by which needle tip proteins control effector secretion is unclear. Here we present data that the P. aeruginosa needle tip protein, PcrV, controls effector secretion by assembling into a functional needle tip complex. PcrV likely does not simply obstruct the secretion channel because the pore‐forming translocator proteins can still be secreted while effector secretion is repressed. This finding suggests that PcrV controls effector secretion by affecting the conformation of the apparatus, shifting it from the default, effector secretion ‘on’ conformation, to the effector secretion ‘off’ conformation. We also present evidence that PcrG, which can bind to PcrV and is also involved in controlling effector export, is cytoplasmic and that the interaction between PcrG and PcrV is not required for effector secretion control by either protein. Taken together, these data allow us to propose a working model for control of effector secretion by PcrG and PcrV.     

Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin

The secretion signal of extracellular metalloprotease B that is secreted without a signal peptide by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi is shown by deletion and gene fusion analyses to be located within the last 40 C-terminal amino acids. Secretion of a peptide containing only this region of the protease requires the same three secretion factors (PrtD, PrtE, and PrtF) that were previously shown to be required for the secretion of the full-length protease. This secretion signal can also be recognized, albeit inefficiently, by the analogous secretion machinery of alpha-hemolysin, another protein with a C-terminal secretion signal that is secreted by some strains of the Gram-negative bacterium Escherichia coli. The secretion signal was fused to an internal 200-amino acid fragment from the sequence of the cytoplasmic protein amylomaltase to promote its specific secretion by the protease secretion pathway. Almost exactly the same sequence as that identified as the protease B secretion signal was also found at the C terminus of metalloprotease C that is also secreted by E. chrysanthemi.     

Characterisation of the slime gland secretion from the peripatus, Euperipatoides kanangrensis (Onychophora: Peripatopsidae)

The Onychophora display a distinctive mechanism of feeding that involves the entanglement of prey in a sticky secretion. This secretion is produced in the slime glands and ejected as adhesive threads from a pair of oral papillae on either side of the head. Biochemical analyses of the secretion reveals it to be a composite material containing protein, sugar, lipid and a surfactant, nonylphenol. The identification of nonylphenol in the secretion is significant in that this is the first report of this compound from a natural source. The proteins are the principal component of the slime and the amino acid composition of the crude secretion suggests the presence of collagen or a ‘collagen-like’ domain. One or more of the high molecular weight proteins are O-glycosylated where the predominant modification is a single N-acetylgalactosamine (GalNAc). This study adds to our understanding of the chemical and biochemical composition of the unusual onycophoran slime gland secretion.     

Secretion processing and activation of Erwinia chrysanthemi proteases

E chrysanthemi, a phytopathogenic enterobacterium, secretes several enzymes into the medium such as pectinases cellulases and proteases. It also produces 3 distinct and antigenically related extracellular proteases. The proteases secretion pathway seems to be distinct from that of the other extracellular enzymes since pleiotropic mutants impaired in cellulase and pectinase secretion are unimpaired in protease secretion. E chrysanthemi proteases B and C secretion occurs without an N-terminal signal peptide and is dependent upon specific secretion functions which are encoded by genes adjacent to the protease structural genes. This secretion pathway might be analogous to the alpha-hemolysin secretion pathway in E coli. Protection against intracellular proteolytic activity is achieved by 2 distinct mechanisms: the proteases are synthesized as inactive precursors with an N-terminal extension of 15 aminoacids (protease B) and 17 aminoacids (protease C) absent in the mature active extracellular enzymes; an intracellular specific protease inhibitor is produced by some E chrysanthemi strains.     

New insights into the distribution of WXG100 protein secretion systems

Protein secretion is an important aspect of bacterial interaction with the environment. The WXG100 secretion system is a poorly understood pathway for the secretion of members of the WXG100 protein family in Firmicutes and Actinobacteria, notably Mycobacteria. This pathway has also been termed the Type VII secretion system but there are semantic problems with this nomenclature. This Perspective reviews the phylum level distribution of WXG100 secretion systems and presents comparative genomic evidence that these systems are present in several Chloroflexi and in some members of the phyla Cyanobacteria, Lentisphaerae, Proteobacteria (notably Helicobacter pylori) and Verrucomicrobiae. These findings have implications for the nomenclature of the WXG100 secretion pathway.     

Feedback control of pancreatic secretion in rats. Role of gastric acid secretion.

Pancreatic secretion in rats is regulated by feedback inhibition of cholecystokinin (CCK) release by proteases in the gut lumen, but little is known about the role of gastric acid in this regulation. This study, carried out on conscious rats with large gastric fistulas (GF) and pancreatic fistulas, shows that diversion of pancreatic juice results in the progressive stimulation of pancreatic secretion only in rats with the GF closed. When the GF was kept open, the diversion resulted in only small increment in pancreatic secretion and this was accompanied by progressive increase in gastric acid outputs. Similar amounts of HCl instilled into the duodenum in rats with the GF open fully reproduced the increase in pancreatic secretion observed after the diversion of pancreatic juice. Pretreatment with omeprazole (15 mumol/kg) to suppress gastric acid secretion or with L-364,718 (5 mumol/kg) to antagonize CCK receptors in the diverted state, resulted in the decline in pancreatic secretion similar to that observed after opening the GF. CCK given s.c. (20-320 pmol/kg) failed to cause any significant rise in the post-diversion pancreatic secretion in rats with the GF closed, but stimulated this secretion dose-dependently when the GF was open. Camostate (6-200 mg/kg) in rats with pancreatic juice returned to the duodenum caused dose-dependent increase in pancreatic secretion, but after opening the GF or after omeprazole this increase was reduced by about 75%. This study provides evidence that gastric acid plays a crucial role in the pancreatic response to diversion of pancreatic juice or inhibition of luminal proteases, and that factors that eliminate gastric acid secretion reduce this response.     

A continuous flow method for the study of lipoprotein lipase secretion in adipose cells

The secretion of lipoprotein lipase has been examined in Ob17 adipose cells. No spontaneous secretion is detected. The activity of the heparin-releasable enzyme shows a first-order process of inactivation. This constant rate of inactivation, coupled with a decreased rate of secretion, prevents any significant determination of enzyme secretion in heparin-containing media. Thus, a perifusion system, with which the rate of enzyme inactivation is minimal and systematic, has been devised and used. The data show that the secretion of a pool of pre-existing lipoprotein lipase molecules is followed by the secretion of newly synthesized enzyme molecules. The results are discussed with respect to the significance of the determinations of the heparin-releasable enzyme in most studies as well as with respect to the intracellular localization of lipoprotein lipase in Ob17 cells.     

Evidence for secretion of 7B2 by A- and B-cells of hamster pancreatic islets.

7B2 is a neuroendocrine protein, and in the pancreatic islets the presence of 7B2 in A- and B-cells was immunohistochemically demonstrated. In order to examine 7B2 secretion by A- and B-cells of pancreatic islets, we prepared isolated hamster pancreatic islet cells as well as an A-cell-rich culture, and studied 7B2 secretion under certain stimulations. 7B2 was secreted by isolated hamster pancreatic islet cells. This secretion was stimulated by theophylline and arginine, but glucose had a weak effect on the 7B2 secretion. Such a response of 7B2 to the stimulations was different from that of insulin or glucagon. 7B2 secretion was also noted in the A-cell-rich culture. These results suggest that 7B2 is secreted by both A- and B-cells of the hamster pancreatic islets and its secretion is regulated under certain conditions.     

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide.     

Glutamine Stimulates Biosynthesis and Secretion of Insulin-like Growth Factor 2 (IGF2), an Autocrine Regulator of Beta Cell Mass and Function

IGF2 is an autocrine ligand for the beta cell IGF1R receptor and GLP-1 increases the activity of this autocrine loop by enhancing IGF1R expression, a mechanism that mediates the trophic effects of GLP-1 on beta cell mass and function. Here, we investigated the regulation of IGF2 biosynthesis and secretion. We showed that glutamine rapidly and strongly induced IGF2 mRNA translation using reporter constructs transduced in MIN6 cells and primary islet cells. This was followed by rapid secretion of IGF2 via the regulated pathway, as revealed by the presence of mature IGF2 in insulin granule fractions and by inhibition of secretion by nimodipine and diazoxide. When maximally stimulated by glutamine, the amount of secreted IGF2 rapidly exceeded its initial intracellular pool and tolbutamide, and high K⁺ increased IGF2 secretion only marginally. This indicates that the intracellular pool of IGF2 is small and that sustained secretion requires de novo synthesis. The stimulatory effect of glutamine necessitates its metabolism but not mTOR activation. Finally, exposure of insulinomas or beta cells to glutamine induced Akt phosphorylation, an effect that was dependent on IGF2 secretion, and reduced cytokine-induced apoptosis. Thus, glutamine controls the activity of the beta cell IGF2/IGF1R autocrine loop by increasing the biosynthesis and secretion of IGF2. This autocrine loop can thus integrate changes in feeding and metabolic state to adapt beta cell mass and function.     

Endogenous opiates inhibit gastric acid secretion induced by central administration of Thyrotropin-Releasing Hormone (TRH)

Thyrotropin-releasing hormone (TRH) administered intraventricularly (ICV) to rats causes a dose-dependent increase in gastric acid secretion over a range of 0.01 μg to 10 μg in the pyloris ligated rat. The maximum increase in gastric acid secretion occurs in the first hour. This effect of TRH is not mediated by its metabolites, histidyl-proline diketopiperazine or pyroglutamyl-histidyl-proline (acid TRH). β-endorphin, D-alanine-methionine-enkephalin and the leucine-enkephalin precursor, dynorphin, all inhibit TRH-induced gastric acid secretion. Bombesin, which reduces basal gastric acid secretion had no effect on TRH-induced secretion.     

细菌三型分泌系统输出蛋白分泌信号研究进展

自提出三型分泌系统的概念以来,相关分子机制的研究让人们对其有了更深入的了解。与依赖信号肽分泌途径形成鲜明对比的是,蛋白通过细菌三型分泌系统分泌或者转运时没有可识别的保守信号序列。近期对三型分泌蛋白的研究发现了多种可以引导其分泌的分泌信号。本文分别介绍了细菌三型分泌系统的种类,分泌系统分泌蛋白的种类,并着重阐述了分泌信号的分子特性及其机制,以期为新型抗菌药物的研发提供新的思路。